Transformasi Tanaman untuk Tujuan Pembungkaman Enzim Pensintesa Etilen Berbasis CRISPR/Cas9 dengan Target Gen ACO6 Exon 1- Genom 12 pada Tanaman Tomat (Solanum Lycopersicum L.)

Abstract

Tomatoes are climacteric fruits with a short shelf life due to the high production of the hormone ethylene during the ripening process. The ACO6 gene (1- aminocyclopropane-1-carboxylic acid oxidase 6) plays a role in the final stage of ethylene biosynthesis, making it a potential target for suppressing ethylene production and extending tomato shelf life. This study aimed to obtain transformant tomato plants with knockout of the ACO6 sgRNA-3 (single guide RNA-3) gene using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) as an alternative genome-editing method for specifically modifying target genes. Transformation was carried out through Agrobacterium tumefaciens strain GV3101 carrying the pRGEB32-ACO6 sgRNA- 3 plasmid construct. Molecular verification via PCR (Polymerase Chain Reaction) using hptII (hygromycin phosphotransferase II) and Cas9 primers showed amplification of specific DNA fragments, indicating successful integration of the construct into the plant genome. The results demonstrated successful transformation through the production of CRISPR/Cas9-based positive transformant tomato plants, confirmed by PCR analysis using hptII and Cas9 primers and visualized through electrophoresis, as indicated by the presence of specific DNA bands in the positive transformants.